Synthesis of biologically active compounds in cells

ABSTRACT

This invention relates to a new method of synthesis of biologically active substances of determined structure directly in the cells of living organisms containing specific RNA or DNA molecules of determined sequence. The method is based on the hybridization of two or more oligomers bound with biologically inactive precursors of biologically active substances to specific RNA or DNA in vivo in the cells of living organisms. After hybridization of the oligomers to RNA or DNA the biologically inactive precursors bound to the 5′ and/or 3′ ends of the oligomers can interact with each other to make biologically active form of the substances. This changing of properties is due to chemical reactions which bind the biologically inactive precursors through a chemical bond into a biologically active form of the whole compound.

This application is a 371 of PCT/IB99/00616 filed Apr. 8, 1999.

Technical field Int. Cl. C07F 9/22; C07F 9/28; C07C 321/00; C07C 323/00U.S. Cl. 560/147: 562/9; 562/10; 562/11 Field of search C07F 9/22; C07F9/28; C07C 321/00; C07C 323/00 References cited U.S. patent documents5,562,350 July 1997 Watanabe et. al., 5,177,198 January 1993 Spielvogelet. al., 5,594,121 January 1994 Froehler et. al., 5,599,922 February1997 Grjasnov et. al., 5,521,302 May 1996 Cook Ph.D. 5,177,064 January1993 Bodor N.S. 5,571,937 November 1996 Kyoichi A. Watanabe.

OTHER REFERENCES

Walder, J. a., et al., (1979), Complementary carrier peptide synthesis:General strategy and implications for prebiotic origin of peptidesynthesis. Proc.Natl.Acad.Sci USA, vol. 76, pp. 51-55.

Ebata K., et al. (1995), Nucleic acids hyridization accompanied withexcimer formation from two pyrene-labeled probes.

Photochemistry and Photobiology, vol. 62(5), pp. 836-839.

Nielsen P. E., (1995), DNA analogues with non phosphodiester backbones.Annu.Rev.Biophys. Biomol.Struct. vol.24, pp. 167-83.

Tam J. P., et al., (1995), Peptide synthesis using unprotected peptidesthrough orthogonal coupling methods.

Proc.Natl.Acad.Sci. USA, vol.92, pp.12485-12489.

Uhlmann G. A. et al., (1990) Antisense Oligonucleotides: A NewTherapeutic principle, Chemical Rev., vol. 90, pp.543-584.

Moser H. E. and Dervan P. B., (1987), Sequence-specific cleavage ofdouble helical DNA by triple helix formation. Science, vol. 238,pp.645-650.

Tulchinsky E. et al., (1992) “Transcriptional analsis of the mts 1 genewith the specific reference to 5′ flanking sequences. Proc.Natl.Acad.SciUSA, vol. 89, pp. 9146-9150.

BACKGROUND ART

The use of oligo(ribo)nucleotides and their analogues as anticancer andantiviruses theraupetic agents was first proposed several years ago.(Uhlmann, 1990) The great number of different modifications of theoligonucleotides and the methods of their use have since been developed.

Two basic interactions between oligonucleotides and nucleic acids areknown (Moser and Dervan, 1987)

1. Watson-Crick base pairing (Duplex structure)

2. Hoogsten base pairing (Triplex structure)

Oligonucleotides can form duplex and/or triplex structures with DNA orRNA of cells and so regulate transcription or translation of genes.

It has been proposed that different substances which can cleave targetnucleic acids or inhibit important cellular enzymes could be coupled tooligomers. The use of such conjugates as therapeutic agents has beendescribed.(U.S. Pat. Nos., 5,177,198; 5,652,350).

Other methods are based on the coupling of different biologically activesubstances, such as toxins, to monoclonal antibodies which can thenrecognise receptors or other structures of cancer cells, or cellsinfected with viruses. Monoclonal antibodies can then specificallyrecognise cancer cells and in this way transport toxins to these cells.But these methods are inefficient due to the high level of nonspecificinteractions between antibodies and other cells, which leads to delivaryof the toxins or other biologically active compounds to the wrong cells.

In 1979 I. M. Klotz and co-authors proposed a method for complementarycarrier peptide synthesis based on a template-directlyed scheme (J. A.Walder et al. 1979) The method proposed the synthesis of peptides on asolid support using unprotected amino acids, and the subsequenthybridization of oligonucleotides on the template. This method wasestablished only for synthesis of peptides in vitro using solid supportsof a different origin, and involved many synthesis steps to obtainpeptides of the determined structure.

M. Masuko and co-authors proposed another method for in vitro detectionof specific nucleic acids by excimer formation from two pyrene-labeledprobes (Ebata, K. et al. 1995).

My invention allows the synthesis of different BACs of determinedstructure directly in living organisms only in cells which have specificRNA or DNA sequences. In this way, BACs will be delivered only to thosecells where specific nucleic acids are produced.

DISCLOSURE OF INVENTION Definitions

“Nulceomonomer”

The term “nucleomonomer” means a “Base” chemically bound to “S”moieties. Nucleomonomers can include nucleotides and nucleosides such asthymine, cytosine, adenine, guanine, diaminopurine, xanthine,hypoxanthine, inosine and uracil. Nucleomonomers can bind each other toform oligomers which can be specifically hybridised to nucleic acids ina sequence and direction specific manner.

The “S” moieties used herein include D-ribose and 2′-deoxy-D-ribose.Sugar moieties can be modified so that hydroxyl groups are replaced witha heteroatom, aliphatic group, halogen, ethers, amines, mercapto,thioethers and other groups. The pentose moiety can be replaced by acyclopentane ring, a hexose, a 6-member morpholino ring; it can beaminoacids analogues coupled to base, bicyclic riboacetal analogues,morpholino carbamates, alkanes, ethers, amines, amides, thioethers,formacetals, ketones, carbamates, ureas, hydroxylamines, sulfamates,sulfamides, sulfones, glycinyl amides other analogues which can replacesugar moieties. Oligomers obtained from the mononucleomers can formstabile duplex and triplex structures with nucleic acids. (Nielsen P. E.1995, U.S. Pat. No. 5,594,121).

“Base”

“Base” (designated as “Ba”) includes natural and modified. purines andpyrimidines such as thymine, cytosine, adenine, guanine, diaminopurine,xanthine, hypoxanthine, inosine, uracil, 2-aminopyridine,4,4-ethanocytosine, 5-methylcytosine, 5-methyluracil, 2-aminopyridineand 8-oxo-N(6)-methyladenine and their analogues. These may include, butare not limited to adding substituents such as —OH, —SH, —SCH(3),—OCH(3), —F, —Cl, —Br, —NH(2), alkyl, groups and others. Also,heterocycles such as triazines are included.

“Nucleotide”

Nucleotide as used herein means a base chemically bound to a sugar orsugar analogues having a phosphate group or phosphate analog.

“Oligomer”

Oligomer means that at least two “nucleomonomers” (defined above) arechemically bound to each other. Oligomers can beoligodeoxyribonucleotides consisting of from 2 to 200 nucleotides,oligoribonucleotides consisting of from 2 to 200 nucleotides, ormixtures of oligodeoxyribonucleotides and oligoribonucleotides. Thenucleomonomers can bind each other through phosphodiester groups,phosphorothioate, phosphorodithioate, alkylphosphonate,boranophosphates, acetals, phosphoroamidate, bicyclic riboacetalanalogues morpholino carbamates, alkanes, ethers, amines, amides,thioethers, formacetals, ketones, carbamates, ureas, hydroxylamines,sulfamates, sulfamides, sulfones, glycinyl amides and other analogueswhich can replace phosphodiester moiety. Oligomers are composed ofmononucleomers or nucleotides. Oligomers can form stable duplexstructures via Watson-Crick base pairing with specific sequences of DNA,RNA, mRNA, rRNA and tRNA in vivo in the cells of living organisms orthey can form stable triplex structures with double stranded DNA ordsRNA in vivo in the cells of living organisms.

“Alkyl”

“Alkyl” as used herein is a straight or branched saturated group havingfrom 1 to 10 carbon atoms. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl and the like.

“Alkenyl”

“Alkenyl” as used herein is a straight- or branched-chainolefinically-unsaturated group having from two to 25 carbon atoms. Thegroups contain from one to three double bounds. Examples include vinyl(—CHdbdCH(2), 1-propenyl (—CHdbdCH—CH(3)), 2-methyl-1-propenyl(—CHdbdC(CH(3))—CH(3)) and the like

“Alkynyl”

“Alkynyl” as used herein is a straight or branchedacetynically-unsaturated groups having from two to 25 carbon atoms. Thegroups contain from one to three triple bounds. Examples include1-alkynyl groups include ethynyl (—CtbdCH), 1-propynyl (—CtbdC—CH(3)),1-butynyl (—CtbdC—CH(2 —CH(3)), 3-methyl-butynyl (—CtbdC—CH(CH(3))—CH(3)), 3,3-dimethyl-butynyl (—CtbdC—C(CH(3))(3)), 1-pentynyl(—CtbdC—CH(2, —CH(2 —CH(3)) and 1,3-pentadiynyl (—CtbdC—CtbdC—CH(3)) andthe like.

“Aryl”

“Aryl” as used herein includes aromatic groups having from 4 to 10carbon atoms. Examples include phenyl, naphtyl and like this.

“Heteroalkyl”

“Heteroalkyl” as used herein is an alkyl group in which 1 to 8 carbonatoms are replaced with N (nitrogen), S (sulfur) or O (oxygen) atoms. Atany carbon atom there can be one to three substituents. The substituentsare selected from: —OH, —SH, —SCH₃, —OCH₃, halogen, —NH₂, —NO₂, —S(O)—,—S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR. Here R is alkyl, alkenyl,aryl, heteroaryl, alkynyl, heterocyclic, carbocyclic and like thisgroups.

“Heteroalkenyl”

“Heteroalkenyl” as used herein is an alkenyl group in which 1 to 8carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen)atoms. At any carbon atom there can be one to three substituents. Thesubstituents are selected from group —OH, —SH, —SCH₃, —OCH₃, halogen,—NH₂, NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR. Here Ris alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic, carbocyclicand like this groups.

“Heteroalkynyl”

“Heteroalkynyl” as used herein is an alkynyl group in which 1 to 8carbon atoms are replaced with N (nitrogen), S (sulfur) or O (oxygen)atoms. At any carbon atom there can be one to three substituents. Thesubstituents are selected from group —OH, —SH, —SCH₃, —OCH₃, halogen,—NH₂, NO₂, —S(O)—, —S(O)(O)′—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR. HereR is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic,carbocyclic and like this groups.

“Heteroaryl”

“Heteroaryl” as used herein means an aromatic radicals comprising from 5to 10 carbon atoms and additionally containing from and to threeheteroatoms in the ring selected from group S, O or N. The examplesinclude but not limited to: furyl, pyrrolyl, imidazolyl, pyridylindolyl, quinolyl, benzyl and the like. One to three carbon atoms ofaromatic group can have substituents selected from —OH, —SH, —SCH₃,—OCH₃, halogen, —NH₂, NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—,—O—P(O)(O)—O—, —NHR, alkyl group. Here R is alkyl, alkenyl, aryl,heteroaryl, alkynyl, heterocyclic, carbocyclic or similar groups.

“Cycloheteroaryl”

“Cycloheteroaryl” as used herein means a group comprising from 5 to 25carbon atoms from one to three aromatic groups which are combined via acarbocyclic or heterocyclic ring. An illustrative radical isfluorenylmethyl. One to two atoms in the ring of aromatic groups can beheteroatoms selected from N, O or S. Any carbon atom of the group canhave substituents selected from —OH, —SH, —SCH₃, —OCH₃, halogen, —NH₂,NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR, alkyl group.Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl, heterocyclic andcarbocyclic and like this groups.

“Carbocyclic”

“Carbocyclic” as used herein designates a saturated or unsaturated ringcomprising from 4 to 8 ring carbon atoms. Carbocyclic rings or groupsinclude cyclopentyl, cyclohexyl and phenyl groups. Any carbon atom ofthe group can have substituents selected from —OH, —SH, —SCH₃, —OCH₃,halogen, —NH₂, NO₂, —S(O)—, —S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—,—NHR, alkyl group. Here R is alkyl, alkenyl, aryl, heteroaryl, alkynyl,heterocyclic and carbocyclic and like this groups.

“Heterocyclic Ring”

“Heterocyclic ring” as used herein is a saturated or unsaturated ringcomprising from 3 to 8 ring atoms. Ring atoms include C atoms and fromone to three N, O or S atoms. Examples include pyrimidinyl, pyrrolinyl,pyridinyl and morpholinyl. At any ring carbon atom there can besubstituents such as —OH, —SH, —SCH₃, —OCH₃, halogen, —NH₂, NO₂, —S(O)—,—S(O)(O)—, —O—S(O)(O)—O—, —O—P(O)(O)—O—, —NHR, alkyl. Where R is alkyl,alkenyl, aryl, heteroaryl, alkynyl, heterocyclic and carbocyclic andlike this groups.

“Hybridization”

“Hybridization” as used herein means the formation of duplex or triplexstructures between oligomers and ssRNA, ssDNA, dsRNA or dsDNA molecules.Duplex structures are based on Watson-Crick base pairing. Triplexstructures are formed through Hoogsteen base interactions. Triplexstructures can be parallel and antiparallel.

The word “halogen” means an atom selected from the group consisting of F(fluorine), Cl (clorine), Br (bromine) and I (iodine)

The word “hydroxyl” means an —OH group:

The word “carboxyl” means an —COOH function.

The word “mercapto” means an —SH function.

The word “amino” means—NH(2) or —NHR. Where R is alkyl, alkenyl, aryl,heteroaryl, heteroalkyl, alkynyl, heterocyclic, carbocyclic and likethis groups.

“Biologically Active Compounds (BACs)”

“Biologically active compound as defined herein include but are notlimited to:

1) biologically active peptides and proteins consisting of naturalaminoacids and their synthetic analogues L, D, or DL configuration atthe alpha carbon atom selected from valine, leucine, alanine, glycine,tyrosine, tryptophan, tryptophan isoleucine, proline, histidine, lysin,glutamic acid, methionine, serine, cysteine, glutamine phenylalanine,methionine sulfoxide, threonine, arginine, aspartic acid, asparagin,phenylglycine, norleucine, norvaline, alpha-aminobutyric acid,O-methylserine, O-ethylserine, S-methylcysteine, S-benzylcysteine,S-ethylcysteine, 5,5,5-trifluoroleucine and hexafluoroleucine. Alsoincluded are other modifications of aminoacids which include but are notlimited to, adding substituents at carbon atoms such as —OH, —SH, —SCH₃,—OCH₃, —F, —Cl, —Br, —NH₂. The peptides can be also glycosylated andphosphorylated.

2) Cellular proteins which include but are not limited to: enzymes, DNApolymerases, RNA polymerases, esterases, lipases, proteases, kinases,transferases, transcription factors, transmembrane proteins, membraneproteins, cyclins, cytoplasmic proteins, nuclear proteins, toxins andlike this.

3) Biologically active RNA such as mRNA, ssRNA, rsRNA and like this.

4) Biologically active alkaloids and their synthetic analogues withadded substituents at carbon atoms such as —OH, —SH, —SCH₃, —OCH₃, —F,—Cl, —Br, —NH₂, alkyl straight and branched.

5) Natural and synthetic organic compounds which can be:

a) inhibitors and activators of the cellular metabolism;

b) cytolitical toxins;

c) neurotoxins;

d) cofactors for cellular enzymes;

e) toxins;

f) inhibitors of the cellular enzymes.

“Precursor(s) of Biologically Active Substances (PBAC(s))”

“Precursors of biologically active compounds (PBACs)” as used herein arebiologically inactive precursors of BACs which can form whole BACs whenbound to each other through chemical moiety(ies) “m” or simultaneouslythrough chemical moieties “m” and “m̂1”. “m” and “m̂1” are selectedindependently from: —S—S—, —O—, —NH—C(O)—, —C(O)—NH—, —C(O)—, —NH—,dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O—, —N═N—.

Biologically active peptides and proteins are synthesized from shorterbiologically inactive peptides. These shorter peptides as used hereinare also biologically inactive precursors of biologically activecompounds.

Biologically active RNAs can be synthesized from biologically inactiveoligoribonucleotides.

“Oligomer-PBAC”

“Oligomer-PBAC” as used herein means a precursor of a BAC (PBAC) whichis chemically bound at the first and/or last mononucleomer at the 3′and/or 5′ ends of the oligomer through the chemical moieties L̂1 and/orL̂2. Chemical moieties L̂1 and L̂2 can be bound directly to a base or to asugar moiety or to sugar moiety analogues or to phosphates or tophosphate analogues,

“Oligomer_(n)-PA_(n)”

“Oligomer_(n)-PA_(n)” as used herein means the precursor of abiologically active protein or RNA which is chemically bound at thefirst and/or last mononucleomer at the 3′ and/or 5′ ends of the oligomerthrough the chemical moieties L̂1 and/or L̂2. n means the ordinal numberof the oligomer of PA. PAs are biologically inactive peptides orbiologically inactive oligoribonucleotides. Wherein n is selected from 2to 300.

a) In Formulas 1 to 4 PBACs are designated as “A” and “B”

A-m-B is equal to a whole BAC “T”

“m” is selected independently from —S—S—, —O—, —NH—C(O)—, —C(O)—NH—,—C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O, —N═N—.

A-O—B is equal to a whole BAC “T”

A-NH—C(O)—B is equal to a whole BAC “T”

A-C(O)—NH—B is equal to a whole BAC “T”

A-C(O)—B is equal to a whole BAC “T”

A-NH—B is equal to a whole BAC “T”

A-dbdN—B is equal to a whole BAC “T”

A-C(O)O—B is equal to a whole BAC “T”

A-C(O)S—B is equal to a whole BAC “T”

A-C(S)S—B is equal to a whole BAC “T”

A-S—S—B is equal to a whole BAC “T”

A-C(S)O—B is equal to a whole BAC “T”

A-N═N—B is equal to a whole BAC “T”

b) Biologically active compounds can be formed through moieties “m” and“m̂1”. “m” and “m̂1” are selected independently from: —S—S—, —O—,—NH—C(O)—, —C(O)—NH—, —C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—,—C(S)S—, —C(S)O, —N═N—, so that

is equal to biologically active compound “T”

a BAC is represented on figure

c) In Formulas 5 to 7, precursors of BACs (PBACs) are designated as“PA_(n)”, where n is selected from 2 to 300. “PA” are peptidesconsisting of from 2 to 100 amino acids or oligoribonucleotidesconsisting of from 2 to 50 ribonucleotides.

{PA₁-m-PA₂-m-PA₃-m- . . . -m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)} isequal to BAC. BACs in this case are proteins or RNAs. Proteins can beenzymes, transcription factors, ligands, signaling proteins,transmembrane proteins, cytolitical toxins, toxins, cytoplasmicproteins, nuclear proteins and the like.

DETAILED DISCLOSURE OF THE INVENTION

This invention relates to the synthesis of biologically active compoundsdirectly in the cells of living organisms. This is achieved by thehybridization of two or more oligomers to cellular RNA or DNA. Theseoligomers are bound to biologically inactive PBACs (oliogmer-PBACs)containing chemically active groups.

BAC can be synthesized only in those cells of living organisms whichhave specific RNA or DNA molecules of a determined sequence.

The principle Formulas of the invention are represented below:

After hybridization of the “Oligomer-PBACs” “A” and “B” to cellular RNA,DNA or dsDNA, the chemically active groups K̂1 and K̂2 of theoligomer-PBACs “A” and “B” interact with each other to form the chemicalmoiety “m”, which combines PBACs “A” and “B” into one active molecule ofbiologically active compound “T”. The degradation of the oligomersand/or linking moieties L̂1 and L̂2 by cellular enzymes or hydrolysisleads to the release of the synthesized BAC “T” directly into thetargeted cells. After hybridization of the oligomer-PBACs to cellularRNA or DNA the distance between the 3′ or 5′ ends of the oligomer A and5′ or 3′ ends of the oligomer B is from 0 to 7 nucleotides of cellularRNA, DNA or dsDNA.

After hybridization of the “oligomer-PBACs” “A” and “B” to cellular RNA,DNA or dsDNA the chemically active group K̂2 of the oligomer-PBAC “B”interacts with the linking moiety L̂1 of the oligomer-PBAC “A” to combinethe PBACs through the chemical moiety “m” into one active molecule ofbiologically active compound “T” with the subsequent release of one PBAC“B” from the oligomer. The degradation of the oligomer and/or linkingmoieties L̂1 by cellular enzymes or hydrolysis leads to the release ofsynthesized BAC “T” directly into the targeted cells.

The chemically active group K̂1 of the oligomer-PBAC A interacts with thelinking moiety L̂2 to combine the PBACs through the chemical moiety “m”into one active molecule of the biologically active compound “T” withthe subsequent release of one PBAC “B” from oligomer “B” and theactivation of the chemical moiety L̂2. After activation, L̂2 interactswith the linking moiety L̂1 to release the biological compound “T” fromthe oligomer directly into targeted cells.

After hybridization, of the “oligomer-PBACs” “A” and “B” to cellularRNA, DNA or dsDNA, the chemically active group K̂2 of the oligomer-PBAC“B” interacts with the linking moiety L̂1 of the oligomer-PBAC “A” tocombine the PBACs through the chemical moiety “m”. At the same time thechemically active group K̂1 of the oligomer-PBAC “A” interacts with thelinking moiety L̂2 of the oligomer-PBAc “B” to form chemical moiety m̂1.Which together with chemical moiety m combines two “Oligomer-PBACs” intoone active molecule of biologically active compound “T”, with therelease of BAC from the oligomer.

After simultaneous hybridization of “Oligomer_(n-1)-PA_(n-1)” and“Oligomer_(n)-PA_(n)” to cellular RNA or DNA, the chemically activegroups K̂1 and K̂2 interact with each other to form the chemical moiety“m” between “Oligomer_(n-1)-PA_(n-1)” and “Oligomer_(n)-PA_(n)”correspondingly; This step is repeated in the cells n-1 times andcombines n-1 times all “PA_(n)”s into one active molecule of thebiologically active compound “PR” which consists of n PA_(n) so thatcompound {“PA”₁-m-“PA”₂-m-“PA”₃-m-“PA”₄-m- . . .-m-“PA_(n-3)”-m-“PA_(n-2)”-m-“PA_(n-1)”-m-“PA_(n)”} is biologicallyactive compound “PR”. The degradation of the oligomers and/or linkingmoieties L̂1 and L̂2 leads to the release of the synthesized BAC “PR”directly into targeted cells of living organism. Here, n is selectedfrom 2 to 2000;

After simultaneous hybridization of “oligomer_(n-1)-PA_(n-1)” and“oligomer_(n)-PA_(n)” to cellular RNA, DNA or dsDNA, the chemicallyactive group K̂1 of “oligomer_(n)-PA_(n)” interacts with the linkingmoiety L̂2 of “oligomer_(n-1)-PA_(n-1)” to bind PA_(n-1) and PA_(n)through chemical moiety “m”. This step is repeated in the cells n-1times and combines n-1 times all PA_(n)s after hybridization of all n“oligomer-PA_(n)”s into one active molecule of the biologically activecompound “PR”, which consists of n PAs so that compound{PA₁-m-PA₂-m-PA₃-m-PA₄-m- . . .-m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)} is equal to the biologicallyactive compound PR. The degradation of the oligomers and/or linkingmoieties L̂1 by cellular enzymes or hydrolysis leads to the release ofthe synthesized BAC PR directly into targeted cells of living organism,here n is selected from to 2000;

After simultaneous hybridization of “Oligomer_(n-1)-PA_(n-1)” and“oligomer_(n)-PA_(n)” to cellular RNA, DNA or dsDNA, the chemicallyactive group K̂1 of “oligomer_(n-1)-PA_(n-1)” interacts with the linkingmoiety L̂2 of “oligomer_(n)-PA_(n)” to bind PA_(n-1) and PA_(n) throughchemical moiety “m”. After interaction of K̂1 with L̂2, L̂2 is chemicallyactivated so that it can interact with linking moiety L̂1 of theoligomer-PA_(n-1), thus destroying the binding of the oligomer_(n-1) toPA_(n-1). This process is repeated n-1 times, so that only whole BAC“PR” comprising from n PA_(n)s {PA₁-m-PA₂-m-PA₃-m-PA₄-m- . . .-m-PA_(n-3)-m-PA_(n-2)-m-PA_(n-1)-m-PA_(n)} is released directly intothe targeted cells of living organisms, here n is selected from 2 to2000.

The chemical moieties in the Formulas 1,2,3,4,5,6 and 7 are as follows:

m is selected independently from: —S—S—, —N(H)C(O)—, —C(O)N(H)—,—C(S)—O—, —C(S)—S—, —O—, —N═N—, —C(S)—, —C(O)—O—, —NH—, —S—;

K̂1 is selected independently from: —NH(2), dbdNH, —OH, —SH, —F, —Cl,—Br, —I, —R̂1-C(X)—X̂1-R̂2;

K̂2 is selected independently from: —NH(2), -dbd-NH, —OH, —SH,—R̂1-C(X)—X̂1-R̂2, —F, —Cl, —Br, —I;

L̂1 is independently: chemical bond, —R̂1-, —R̂1-O—S—R-̂2-, —R̂1-S—O—R-̂2-,—R̂1-S—S—R̂2-, —R̂1S—N(H)—R̂2-, —R̂1-N(H)—S—R̂2-, —R̂1-O—N(H)—R̂2-,—R̂1-N(H)—O—R̂2-, —R̂1-C(X)—X—R̂2-;

L̂2 is independently: chemical bond, —R̂1, —R̂1-O—S—R̂2-, —R̂1-S—O—R̂2-,—R̂1-S—S—R̂2-, —R̂1-S—N(H)—R̂2-, —R̂1-N(H)—S—R̂2-, —R̂1-O—N(H)—R̂2-,—R̂1-N(H)—O—R̂2-, —R̂1-C(X)—X̂1-R̂2-, —R̂1-X—C(X)—X—C(X)—X—R̂2-;

R-̂1 is independently: chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X̂1-P(X)(X)—X̂1, —S(O)—, —S(O)(O)—,—X̂1-S(X)(X)—X̂1-, —C(O)—, —N(H)—, —N═N—, —X̂1P(X)(X)—X̂1-, —X̂1-P(X)(X)—X̂1-.

P(X)(X)—X̂1, —X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —C(S)—, any suitablelinking group;

R̂2 is independently chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X̂1-P(X)(X)—X̂1, —S(O)—, —S(O)(O)—,—X̂1-S(X)(X)—X-̂1-, —C(O)—, —N(H)—, —N═N—, —X̂1-P(X)(X)—X̂1-,—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —C(S)—,any suitable linking group;

X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl;

X̂1 is independently S, O, NH, Se, alkyl, alkenyl, alkynyl.

In Formulas 1,2,3,4,5,6 and 7 the linking moieties L̂1 and L̂2 are boundto the first and/or last mononucleomers of the oligomers at their sugaror phosphate moiety, or directly to base, or to sugar moiety analogues,or to phosphate moiety analogues, or to base analogues.

All the described schemes demonstrate that BACs can not be synthesizedin non-targeted cells because the molar concentration of the chemicallyactive groups is too low, and without hybridization of theoligomer-PBACs to the template, specific reactions can not occur. Afterhybridization of the oligomer-PBACs to a specific template, theconcentration of the chemically active groups is sufficient for thechemical reaction between the chemical groups of PBACs to occur. Thereaction leads to chemical bond formation between PBACs and subsequentformation of a whole BAC. The degradation of the oligomers and/orlinking moieties of the oligomers with PBACs leads to the release ofBACS directly into targeted cells. To synthesise directly incellsbiologically active polymers such as proteins and RNAs ofdetermined structure more than two PBACs are used. PBACs for synthesisof proteins or RNAs are designated as PA_(n). PA_(n) are peptides oroligoribonucleotides. The mechanisms of the interaction of such PBACsare the same as in the synthesis of small biologically active compounds.The difference is that the PBACs (with the exception of the first andlast PBACs) are bound simultaneously to the 5′ and 3′ ends of theoligomers so that the direction of synthesis of the biologically activeprotein or RNA can be determined.

Possible functions of BACs synthesized by proposed methods are: 1)Killing of cells, 2) Stimulation of the metabolism of cells 3) Blockingof important ion channels such as Na⁺, K⁺, Ca⁺⁺ and other ion channels,in order to inhibit signal transmissions. BACs can be proteins,peptides, alkaloids, synthetic organic compounds. They can be cleavedinto two or more precursors called PBACs. After interaction between thechemical groups of PBACs, whole BAC is formed through the moiety “m”.

a) In Formula 1,2,3 and 4 PBACs are designated as “A” and

A-m-B is equal to a whole BAC “T”

“m” is selected independently from —S—S—, —O—, —NH—C(O)—, —C(O)—NH—,—C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—, —C(S)S—, —C(S)O—, —N═N—.

A-O—B is equal to a whole BAC “T”

A-NH—C(O)—B is equal to a whole BAC “T”

A-C(O)—NH—B is equal to a whole BAC “T”

A-C(O)—B is equal to a whole BAC “T”

A-NH—B is equal to a whole BAC “T”

A-dbdN—B is equal to a whole BAC “T”

A-C(O)O—B is equal to a whole BAC “T”

A-C(O)S—B is equal to a whole BAC “T”

A-C(S)S—B is equal to a whole BAC “T”

A-S—S—B is equal to a whole BAC “T”

A-C(S)O—B is equal to a whole BAC “T”

A-N═N—B is equal to a whole BAC “T”

b) A biologically active compound can be formed through the moieties “m”and “m̂1”. “m” and “m̂1” are selected independently from: —S—S—, —O—,—NH—C(O)—, —C(O)—NH—, —C(O)—, —NH—, dbdN—, —C(O)O—, —C(O)S—, —S—,—C(S)S—, —C(S)O—, —N═N—, so that

is equal to biologically active compound “T”

This kind of interaction is represented in FIG. 4.

c) In Formulas 5, 6 and 7, precursors of BACs (PBACS) are designated as“PA_(n)”, where n is selected from 2 to 2000. “PA” are peptides oroligoribonucleotides consisting of from 2 to 100 amino acids. n is theordinal number of PA in a series of PAs and designates the sequence ofbinding of PAs to each other.

{“PA₁”-m-“PA₂”-m-“PA₃”-m- . . .-m-“PA_(n-3)”-m-“PA_(n-2)”-m-“PA_(n-1)”-m-“PA_(n)”} is equal to BAC“PR”. BACs “PR” in this case are proteins or RNAs. Proteins can becellular proteins, enzymes, transcription factors, ligands, signallingproteins, transmembrane proteins, cytolitical toxins, cytoplasmic andnuclear proteins and the like. RNAs are selected from mRNA, rsRNA andthe like.

BRIEF DESCRIPTION OF DRAWNINGS

FIG. 1 Synthesis of the Toxin Daphnoretin

Toxin Daphnoretin is cleaved into two precursors. After simultaneoushybridization to cellular RNA of the oligomers bound to the daphoretin'sprecursors, the chemically active hydroxyl group of daphnoretin'sprecursor “A” interacts with the chemically active Cl group of precursor“B” to form a chemical bond between the two daphnoretin precursors. Thedegradation of the linking moieties and/or oligomers leads to therelease of the biologically active molecule directly into targetedcells.

FIG. 2 Synthesis of the Neurotoxin Peptide

Neurotoxin is cleaved into two shorter, biologically inactive peptides.After hybridization to cellular RNA or DNA, the chemically active NH₂group of peptide “A” interacts with the linking moiety —C(O)—O-L̂2,forming a peptidyl bond. After the peptidyl bonds formation, thechemically active group —SH of peptide “B” interacts with the linkingmoiety L̂1-S—S— which binds peptide “A” with oligomer “A”. After thisinteraction, an —S—S— bound between the two cysteins is formed and thebiologically active neurotoxin is released into targeted cells.Aminoacids are designated as italicised letters in one letter code.

FIG. 3 The Synthesis of the Toxin Tulopsoid A

Toxin tulopsoid A is cleaved into two precursors. After simultaneoushybridization to cellular RNA of the oligomers bound to the tulopsoid Aprecursors chemically active hydroxyl group of the oligomer-PBAC “A”interacts with the —CH₂—S—C(O)— linking moiety to form a chemical bondwith tulopsoid's precursor “B”, releasing precursor “B” from oligomer 2.The activated —CH₂—SH moiety interacts with the linking moiety —S—O—,releasing the whole tulopsoid A from oligomer 1.

FIG. 4 Synthesis of the Toxin Amanitin

Toxin-amanitin is a strong inhibitor of transcription. It can be cleavedinto two inactive precursors which can be used to synthesise the wholemolecule of amanitin. After hybridization of all oligomers bound withthe amanitin's precursors to cellular RNA or DNA, free amino group ofamanitin's precursor “A” can interact with the carboxyl group —C(O)—S-L̂2to form a peptidyl bond and to release amanitin's precursor “B” fromoligomer 2. The linking moiety of amanitin's precursor “A” to theoligomer 1 is semistabile. The release of precursor “A” from theoligomer 1 is performed due to action of the activated —SH group on thelinking moiety —C(O)—O—S-L̂1. Oligomers 3 and 4 bound with the amanitin'sprecursors “A” and “B” are hybridized on the same molecule of RNA orDNA. The amino group of amanitin's precursor “B” interacts with thecarboxyl group —C(O)—S-L̂1 to form a peptidyl bond, releasing amanitin'sprecursor “A” from the oligomer 3. The linking moiety of amanitin'sprecursor “B” to the oligomer 4 is semistabile. The release of precursor“B” from the oligomer 4 is performed due to action of the activated —SHgroup on the linking moiety —C(O)—O—S-L̂2.

FIG. 5 Synthesis of the Toxin D-actinomicin

Toxin D-actinomicin is cleaved into two precursors. After simultaneoushybridization of two oligomer-PBACs to cellular RNA or DNA chemicallyactive amino and halogen groups of precursor “A” interact with thechemically active halogen and hydroxyl groups of D-actinomicin'sprecursor “B” respectively to form two chemical bonds between theprecursors.

FIG. 6 Synthesis of the Toxin Ochratoxin A

Toxin ochratoxin A is cleaved into two precursors which are bound tooligomers. After simultaneous hybridization of the oligomer-PBACs tocellular RNA or DNA, the chemically active amino group of precursor “B”interacts with the moiety C(O)—O— which links precursor “A” witholigomer A, to form a chemical bond between the two ochratoxinprecursors. After oligomer or linking moiety degradation in the cellsthe whole biologically active molecule of Ochratoxin A is released intothe targeted cells.

FIG. 7 Synthesis of the Toxin Ergotamin

Toxin ergotamin is cleaved into two precursors which are bound tooligomers. After simultaneous hybridization of the oligomer-PBACs tocellular RNA or DNA, the chemically active amino group of precursor “B”interacts with the moiety C(O)—O— which binds precursor “A” witholigomer “A”, to form a chemical bond between the two ergotaminprecursors. After degradation of the oligomers, RNA, or DNA in thecells, the whole biologically active molecule of ergotamin is releasedinto the targeted cells.

FIG. 8. Synthesis of Proteins

The synthesis of a biologically active protein of n peptides.

Peptides are bound to oligomers simultaneously at their amino andcarboxy ends, with the exception of the first peptide which is bound tothe oligomer at its carboxy end, and the last peptide which is bound tothe oligomer at its amino terminus. Two oligomers bound to peptides(oligomer-PAs) are hybridized simultaneously to specific RNA or DNAmolecules, the distance from each other between 0 and 10 nucleotides ofcellular RNA or DNA. After hybridization, the amino group of theoligomer-PA_(n) interacts with the -L̂2-S—C(O)— linking moiety to form apeptidyl bond between peptide “_(n-1)” and peptide “n”. Thepeptide_(n-1) is released from the oligomer_(n-1) at its carboxyterminus. The activated -L̂2-SH group interacts then with the linkingmoieties —O—S-L̂1 and —O—NH-L̂1 which bind peptides_(n) at theirN-terminus with oligomers_(n). After hybridization of all n oligomer-PAsthe process is repeated n-1 times to bind all n peptides into onebiologically active protein. Linking of the peptides at the N-terminuswith oligomers is performed by aminoacids which have hydroxyl group suchas serine, threonine and tyrosine.

FIG. 9. Synthesis of Proteins

The same process is shown as in FIG. 8, but this time the peptides arebound at their N terminus to oligomers through aminoacids with aminoand-mercapto groups, for example cysteine, arginine, asparagine,glutamine and lysine. The activated -L̂2-SH group can interact with thelinking groups such as —S—S-L̂1, —S—NH-L̂1 to form -L̂2-S—S-L̂1-,-L̂2-S—NH-L̂1 moieties and to release peptides from oligomers at their Nterminus.

FIG. 10. Synthesis of RNA

In this figure “PA_(n)” are oligoribonucleotides comprising from 3 to300 nucleotides.

n in “PA_(n)” means the ordinal number in a series ofoligoribonucleotides used in the synthesis of whole RNA, where n isselected from 2 to 1000.

PA₁ couples with PA₂ through the chemical moiety —O—, then in turnPA₁-m-PA₂ couples with PA₃ through chemical moiety —O—, thenPA₁-m-PA₂-m-PA₃ couples with PA₄ through chemical moiety —O— and so onuntil the last “n”th oligoribonucleotide is bound, forming the wholebiologically active RNA.

The chemical moieties in FIGS. from 1 to 10 are as follows:

m is selected independently from: —S—S—, —N(H)C(O)—, —C(O)N(H)—,—C(S)—O—, —C(S)—S—, —O—, —N═N—, —C(S)—, —C(O)—O—, —NH—, —S—;

K̂1 is selected independently from: —NH(2), dbdNH, —OH, —SH, —S, —Cl,—Br, —I, —R̂1-C(X)—X̂1-R̂2;

K̂2 is selected independently from: —NH(2), -dbd—NH, —OH, —SH,—R̂1-C(X)—X̂1-R̂2, —F, —Cl, —Br, —I;

L̂1 is independently: chemical bond, —R̂1-, —R̂1-O—S—R̂2-, —R̂1-S—O—R̂2-,—R̂1-S—S—R̂2-, —R̂1-S—N(H)—R̂2-, —R̂1-N(H)—S—R̂2-, —R̂1-O—N(H)-—R̂2-,—R̂1-N(H)—O—R̂2-, —R̂1-C(X)—X—R̂2-;

L̂2 is independently: chemical bond, —R̂1-, —R̂1-O—S—R̂2-, —R̂1-S—O—R̂2-,—R̂1-S—S—R̂2-, —R̂1-S—N(H)—R̂2-, —R̂1-N(H)—S—R̂2-, -R̂1-O—N(H)—R̂2-,—R̂1-N(H)—O—R̂2-, —R̂1-C(X)—X̂1-R̂2-, —R̂1-X—C(X)—X—C(X)—X—R̂2-;

R̂1 is independently: chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X̂1-P(X)(X)—X̂1, —S(O)—, —S(O)(O)—,—X̂1-S(X)(X)—X̂1-, —C(O)—, —N(H)—, —N═N—, —X̂1-P(X)(X)—X̂1-,—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —C(S)—,any suitable linking group;

R̂2 is independently chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X̂1-P(X)(X)—X̂1, —S(O)—, —S(O)(O)—,—X̂1-S(X)(X)—X̂1-, —C(O)—, —N(H)—, —N═N—, —X̂1-P(X)(X)—X̂1-,—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —C(S)—,any suitable linking group;

X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl;

X̂1 is independently S, O, NH, Se, alkyl, alkenyl, alkynyl.

BEST MODE FOR CARRYING OUT THE INVENTION The Synthesis of DifferentToxins and Alkaloids Directly Into Targeted Cells EXAMPLE 1 TheSynthesis of the Toxin Alpha Amanitin

The amanitin is a toxin present in mushrooms. It acts as a very stronginhibitor of transcription in eucaryotic cells, and is therefore verystrong toxin.

The synthesis of alpha-amanitin is represented in FIG. 1 The structureof the toxin is a cyclic peptide with modified amino acids. The moleculeof alpha-amanitin can be cleaved into two inactive precursors, which arebound to 4 oligomers through linking moieties L̂1 and L̂2, designated inFIG. 1. After hybridization of all oligomers to the same molecule of RNAthe synthesis of toxin amanitin is occured.

EXAMPLE 2 The Synthesis of Biologically Active Peptides

The synthesis of BACs consisting of amino acids makes possible thesynthesis of practically any peptide. These peptides can be involved ina wide variety of processes. The specific synthesis will occur only inthe cells where the specific sequences are represented.

The synthesis of peptides such as endorphins or toxins which block Na,K, Ca channels can be performed directly on specific RNA or DNAsequences. These peptides can act as agents stimulating cells of thenervous system, or as analgesic agents. To date, the number of knownbiologically active peptides is enormous. The peptides can besynthesized from natural aminoacids as well as from synthetic aminoacids of D or L conformations.

The synthesis of neurotoxin is represented in FIG. 2.

EXAMPLE 3 The Synthesis of the Toxin Tulopsoid A

Toxin tulopsoid A is an alkaloid and is a strong cytolitical toxin.

Toxin tulopsoid A is cleaved into two precursors. The chemically activehydroxyl group of precursor “A” can interact after hybridization withthe —CH₂—S—C(O)— moiety to form a chemical bond with tulopsoid'sprecursor “B”, with the release of precursor “B” from the oligomer. Theactivated —CH₂—SH moiety interacts with the linking moiety —S—O—,releasing the whole tulopsoid from oligomer (FIG. 3).

EXAMPLE 4 The Synthesis of the Toxin Daphnoretin

Toxin daphnoretin is an alkaloid and is a strong cytolitical toxin.

Toxin Daphnoretin is cleaved into two precursors. After simultaneoushybridization of the oligomers coupled to the daphnoretin's precursorsthe chemically active hydroxyl group of daphnoretin's precursor “A”interacts with the chemically active Cl group of precursor “B” to formchemically bond between daphnoretin's precursors. The degradation of theoligomers or linking groups leads to the release of the biologicallyactive molecule directly into targeted cells (FIG. 4).

EXAMPLE 5 The Synthesis of the Toxin D-actinomicin

Toxin D-actinomicin is an alkaloid and is a strong cytolitical toxin.

Toxin D-actinomicin is cleaved into two precursors. After hybridizationof two oligomers to cellular RNA or DNA, the chemically active groupsamino and halogen of precursor “A” interact with the chemically activegroups halogen and hydroxyl respectively of D-actinomicin's precursor“B” to form two chemical bonds between the precursors (FIG. 5.).

EXAMPLE 6 The Synthesis of the Toxin Ochratoxin A

Toxin ochratoxin A is an alkaloid and is a strong cytolitical toxin.

Toxin ochratoxin A is cleaved into two precursors bound to oligomers.After hybridization of the oligomers to cellular RNA or DNA, thechemically active amino group of the precursor “B” interacts with themoiety —O—C(O) of precursor “A” to form a chemical bond between the twoochratoxin precursors. After the degradation of the oligomers or linkingmoieties in the cells, whole, biologically active molecules ofOchratoxin A will be released into targeted cells (FIG. 6.).

EXAMPLE 7 The Synthesis of the Toxin Ergotamin

Toxin ergotamin is an alkaloid and is a strong cytolitical toxin.

Toxin ergotamin is cleaved into two precursors which are bound tooligomers. After hybridization of the oligomers to cellular RNA or DNA,the chemically active amino group of precursor “B” interacts with moiety—O—C(O) of precursor “A” to form a chemical bond between the twoergotamin precursors. After degradation of the oligomers or linkingmoieties in the cells, whole, biologically active molecules of ergotaminwill be released into the targeted cells.

By using more than two oligonucleotides bound at their 5′,3′ ends toprecursors of biologically active compounds, higher concentration levelof the biologically active substances can be achieved into targetedcells.

O11, O12, O13 are oligomers 1,2,3 which at their 3′ and 5′ ends arebound to precursors of biologically active substances.

Such linking can also prevent oligonucleotides from exonucleasedegradation and constabilise their activity in cells. In any case, theproducts of the degradation of the peptides and oligonucleotides formedfrom natural aminoacids and nucleotides are not toxic, and can be usedby cells without elimination from the organism or toxic effects on otherhealthy cells.

All the toxins described can be used for the synthesis of toxins incells infected by viruses, using the hybridization of the oligomers todouble stranded DNA. In U.S. Pat. No. 5,571,937 the homopurine sequencesof HIV 1 were found.

One such sequence is 5′-GMGGMTAGMGMGMGGTGGAGAGAGAGA-3′ (seq ID NO 43U.S. Pat. No. 5,571,937). Using two oligomers: (A-5′-GMGGMTAGAAGMG-3′)(SEQ ID No. 1) and (B-5′-MGMGGTGGAGAGAGAGA-3′) (SEQ ID No. 2) boundthrough linking moieties L 1 and L 2 to PBACs, synthesis of thecorresponding BACs directly in human cells infected by HIV1 can beachieved. The toxin will be synthesized only in thouse cells infected byHIV1. Other healthy cells will be not killed by synthesized toxin.

The Synthesis of Proteins

The synthesis of protein can be performed according to the schemedesignated in Formulas 5, 6 and 7 and in FIGS. 8, 9.

Relatively small molecules can be used to synthesize the whole activeproteins in any tissue of a living organism. These small molecules caneasily penetrate the blood brain barrier, or enter other tissues. Thedegradation products of such compounds can be used as nutrients forother cells. They are also not toxic to other cells where specific RNAsare not present, in the case where oligomers areoligoribo(deoxy)nucleotides. The synthesis of whole proteins of 50 kDacan be performed on one template 300-500 nucleotides in length usingoligomers of the length 10-50 nucleomonomers bound to peptidesconsisting of 2-30 amino acids. Only 10-20 such PBACs are necessary tosynthesise a protein of molecular weight 50 kDa. Theoretically, it ispossible to synthesise the proteins of any molecular mass. The number ofoligomer-PAs can vary from 1 to 1000, but the efficiency of synthesis oflarge proteins is very low and depends on the velocity of the reactionand the degradation of the oligomer-PAs in the living cells.

By this method, synthesized proteins can be modified later in the cellsby cellular enzymes to achieve the biologically active form of theprotein.

The method allows the synthesis of specific proteins only in thousecells in which the proteins are needed. Any type of proteins can besynthesized by this method. These proteins can be involved in cellularmetabolism, transcription regulation, enzymatic reactions, translationregulation, cells division or apoptosis.

The mechanism allows the synthesis of any protein directly into targetedcells. The synthesized proteins could inhibit a cell's growth ordivision, or could stimulate division and metabolism of cells wherespecific RNAs are expressed. By the method described, it is possible tosynthesise not only one protein, but many different proteins in theselected cells. These proteins could change even the differentiation ofthe targeted cells. The targeted cells can be somatic cells of livingorganisms, tumour cells, cells of different tissues, bacterial cells orcells infected by viruses.

EXAMPLE 8 Synthesis of the Tumour Suppresser p53

The synthesis is performed according to Formula 6. In the example below,the peptides from PA₂ to PA₁₄ are bound at their NH₂ end to the linkingmoiety L̂2 through the OH group of amino acids serine or threonine. Thelinking moiety L̂2 is bound to the phosphate or sugar moiety of thenucleotides localised at the 5′ end of the corresponding oligomers. Theamino acids at the COOH ends of the peptides are bound to the oligomerthrough acyl moieties (L̂1) bound to the 3′ OH group of sugar moiety ofthe nucleotide localised at 3′ end. After hybridization to specificcellular RNA, the NH₂ group of the oligomer_(n)-PA_(n) interacts withthe linking acyl group of the oligomer_(n-1)-PA_(n-1) to form a peptidylbond between two oligomer-PAs. The whole P53 protein can be synthesizedusing only 14 oligomer-PAs and a 250 nucleotide long region of RNA forhybridization to the oligomer-PAs.

PA₁, PA₂, PA₃, PA₄, PA₅, PA₆, PA₇, PA₈, PA₉, PA₁₀, PA₁₁, PA₁₂, PA₁₃ andPA₁₄ are the peptides which are bound to the oligomers. The sequences ofthe peptides are represented below.

PA₁—MEEPQSOPSV EPPLSQETFS DLWKLLPENN VL (SEQ ID No. 3)

PA₂—SPLPSQAH DDLMLSPDDI EQWF (SEQ ID No. 4)

PA₃—TEDPGPDEAP RHPEAAPRVA PAPMP (SEQ ID No. 5)

PA₄—TPAAPAPAPS WPLSSSVPSQ KTYQG (SEQ ID No. 6)

PA₅—SYGFRLGFLHS GTAKSVTCRY (SEQ ID No. 7)

PA₆—SPAL NKMFCQLAKT CPVQLWVDSTPPPG (SEQ ID No. 8)

PA₇—TRVRAM AIYKQSQHMT EVVRRCPHHE (SEQ ID No. 9)

PA₈—TCSDSDGLAP PQHLIRVEGN LRVEYLDDRN (SEQ ID No. 10)

PA₉—TFRHSVVVPY EPPEVGSDCT TIHYNYMCNS (SEQ ID No. 11)

PA₁₀—SCMGGMNRRP ILTIITLEDS SGNLLGRN (SEQ ID No. 12)

PA₁₁—SFEVRVCACPGR DRRTEEENLR KKGEPHHELPPG (SEQ ID No. 13)

PA₁₂—STKRALPN NTSSSPQPKK KPLDGEYF (SEQ ID No. 14)

PA₁₃—TLQIRGRERFEM FRELNEALEL KDAQAGKEPGG (SEQ ID No. 15)

PA₁₄—SRAHSSHLK SKKGQSTSRH KKLMFKTEGP DSD (SEQ ID No. 16)

Aminoacids are designated in bold/italicised one letter code.

A—alanine, R—arginine, N—asparagine, D—aspartic acid, C—cysteine,Q—glutamine, E—glutamic acids, G—glycine, H—histidine, I—isoleucine,L—leucine, K—lysine, M—methionine, F—phenylalanine, P—proline, S—serine,T—threonine, W—tryptophan, Y—tyrosine, V—valine.

The tyrosine in PA₇ can be chemically phosphorylated. In this way analready active form of the protein can be synthesized directly in thecells. It is possible to include any modification at any amino acid ofthe PAs.

(SEQ ID NO. 17) oligomer 1 5′-cccaatccctcttgcaactga-3′ (SEQ ID NO. 18)oligomer 2 5′-attctactacaagtctgccctt-3′ (SEQ ID NO. 19) oligomer 35′-ttgtgaccggctccactg-3′ (SEQ ID NO. 20) oligomer 45′-taccttggtacttctctaa-33′ (SEQ ID NO. 21) oligomer 55′-atgccatattagcccatcaga-33′ (SEQ ID NO. 22) oligomer 65′-ccaagcattctgtccctccttt-3′ (SEQ ID NO. 23) oligomer 75′-tccggtccggagcacca-3′ (SEQ ID NO. 24) oligomer 85′-gccatgacctgtatgttaca-3′ (SEQ ID NO. 25) oligomer 95′-ggtgtgggaaagttagcggg-3′ (SEQ ID NO. 26) oligomer 105′-gcgaattccaaatgattttaa-33′ (SEQ ID NO. 27) oligomer 115′-aatgtgaacatgaataa-33′ (SEQ ID NO. 28) oligomer 125′-agagtgggatacagcatctata-3′ (SEQ ID NO. 29) oligomer 135′-acaaaaccattccactctgatt-3′ (SEQ ID NO. 30) oligomer 145′-ttggaaaaactgtgaaaaa-3′

All oligomers herein are oligonucleotides antiparallel to the humanplasminogen antigen activator mRNA. After hybridization of theoligomer-PAs to the RNA, the distance between the 3′ ends of theoligomer_(n-1) and the 5′ ends of the oligomer_(n) is equal to 0nucleotides of plasminogen antigen activator mRNA. n as used herein isfrom 1 to 14.

Oligomer1-PA₁ is H₂N-MEEPOSDPSVEPPLSQETFSDLWKLLPENNVL (SEQ ID No. 3)                                  L{circumflex over ( )}15′-cccaatcoctcttgcaactga-3′ (SEQ ID No. 17) Oligomer₂-PA₂ isH₂N-SPLPSOAMDDLMLSPDDIEQWF (SEQ ID No. 4)   L{circumflex over( )}2                   L{circumflex over ( )}15′-attctactacaagtctgccctt-3′ (SEQ ID No. 18) Oligomer₃-PA₃ isH₂N-TEDPGPDEAPRMPEAAPRVAPAPMP (SEQ ID No. 5)   L{circumflex over( )}2                       L{circumflex over ( )}15′-ttgtgaccggctccactg-3′ (SEQ ID No. 19) Oligomer₄-PA₄ isH₂N-TPAAPAPAPSWPLSSSVPSQ- KTYQG (SEQ ID No. 6)    L{circumflex over( )}2                        L{circumflex over ( )}15′-taccttqgtacttctctaa-3′ (SEQ ID No. 20) Oligomer₅-PA₅ isH₂N-SYGFRLGFLHSGTAKSVTCTY (SEQ ID No. 7)    L{circumflex over( )}2                 L{circumflex over ( )}15′-atgccatattagcccatcaga-3′ (SEQ ID No. 21) Oligomer₆-PA₆ isH₂N-SPALNKMFCQLAKTCPVQLWVDSTPPPG (SEQ ID No. 8)    L{circumflex over( )}2                       L{circumflex over ( )}15′-ccaagcattctgtccctccttt-3′ (SEQ ID No. 22) Oligomer₇-PA₇ isH₂N-TRVRAMAIYKQSQHMTEVVRRCPHHE (SEQ ID No. 9)    L{circumflex over( )}2                      L{circumflex over ( )}15′-tccggtccggagcacca-3′ (SEQ ID No. 23) Oligomer₈-PA₈ isH₂N-TCSDSDGLAPPQHLIRVEGNLRVEYLDDRN (SEQ ID No. 10)   L{circumflex over( )}2                          L{circumflex over ( )}15′-gccatgacctgtatgttaca-3′ (SEQ ID No. 24) Oligomer₉-PA₉ isH₂N-TFRHSVVVPYEPPEVGSDCTTIHYNYMCN (SEQ ID No. 11)    L{circumflex over( )}2                         L{circumflex over ( )}15′-ggtgtgggaaagttagcggg-3′ (SEQ ID No. 25) Oligomer₁₀-PA₁₀ isH₂N-SSCMGGMNRRPILTIITLEDSSGNLLGRN (SEQ ID No. 12)    L{circumflex over( )}2                         L{circumflex over ( )}15′-gcgaattccaaatgattttaa-3′ (SEQ ID No. 26) Oligomer₁₁-PA₁₁ isH₂N-SFEVRVCACPGRDRRTEEENLRKKGEPHHELPPG (SEQ ID No. 13)  L{circumflexover ( )}2                              L{circumflex over ( )}15′-aatgtgaacatgaataa-3′ (SEQ ID No. 27) Oligomer₁₂-PA₁₂ isH₂N-STKRALPNNTSSSPQPKKKPLDGEYF (SEQ ID No. 14)      L{circumflex over( )}2                    L{circumflex over ( )}15′-agagtgggatacagcatctata-3′ (SEQ ID No. 28) Oligomer₁₃-PA₁₃ isH₂N-TLQIRGRERFEMFRELNEALELKDAQAGKEPGG (SEQ ID No. 15)    L{circumflexover ( )}2                            L{circumflex over ( )}15′-acaaaaccattccactctgatt-3′ (SEQ ID No. 29) Oligomer₁₄-PA₁₄ isH₂N-SRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID No. 16)    L{circumflexover ( )}2 5′-ttggaaaaactgtgaaaaa-3′ (SEQ ID No. 30)

The oligomer_(n)-PA_(n) (n is selected from 1 to 14) are peptideschemically bound to oligomers which can form stable duplex structurewith the plasminogen antigen activator mRNA expressed in human ovariantumour cells. Using the plasminogen antigen activator mRNA it ispossible to synthesize any other protein or small BAC. All theseproteins or BACs will be synthesized only in those cells where the humanplasminogen activator mRNA is expressed. In the case of the humanplasminogen activator mRNA, the synthesis of the protein or BAC willoccur only in ovarian tumour cells. Oligomer 1 at its 3′ end is bound tothe “C” end of the peptide PA₁ of p53 through the linking moiety L̂1.Oligomers 2 to 13 are bound at their 5′ and 3′ ends to peptides PA₂ toPA₁₃ at their “N” and “C” ends respectively, through the linkingmoieties L̂2 and L̂1. Oligomer14 at it's 5′ end is bound to the “N” end ofthe peptide PA₁₄ of p53 through the linking moiety L̂2. The firstmethionine of PA₁ is formylated, and the amino end of peptide₁ is notbound to Oligomer₁. The last amino acid at the carboxyl end of PA₁₄ isnot bound to Oligomer₁₄. Only 14 peptides chemically bound to 14oligomers are required to synthesize p53 tumour suppresser specificallyin the cells of the ovarian tumour. In any type of tumour cell RNAsspecific to this cell type are expressed. By this method, it is possibleto synthesise any protein or BACs described above on these RNAs.

The 14 Oligomer-PAs are hybridized on the mRNA in such a manner that the3′ end of the oligomer₁-PA₁ is located at a distance from the 5′ end ofthe oligomer₂-PA₂ which is equal to 0 nucleotides of the plasminogenantigen activator mRNA. The distance between the 5′ end of theOligomer₃-PA₃ and the 3′ end of the Oligomer₂-PA₂ is equal to 0nucleotides of the plasminogen antigen activator mRNA. The distancebetween the 5′ end of the Oligomer₄-PA₄ and the 3′ end of theoligomer₃-PA₃ is equal to 0 nucleotides of the plasminogen antigenactivator mRNA etc. In other words, after hybridization of theoligomer-PAs to the plasminogen antigen activator mRNA, the distancebetween the 3′ end of the oligomer_(n-1)-PA_(n-1) and the 5′ end of theOligomer_(n)-PA_(n) is equal to 0 nucleotides of the plasminogen antigenactivator mRNA.

After the degradation of the oligomers and/or linking moieties, thesynthesized protein p53 is released into the determined cells.

{H₂N-PA₁-C(O)NH-PA₂-C(O)NH-PA₃-C(O)NH-PA₄-C(O)NH-PA₅-C(O)NH-PA₆--C(O)NH-PA₇-C(O)NH-PA₈-C(O)NH-PA₉-C(O)NH-PA₁₀-C(O)NH-PA₁₁-C(O)NH--PA₁₂-C(O)NH-PA₁₃-C(O)NH-PA₁₄-COOH} is biologically activeprotein—tumour suppresser p53. The yield of synthesis in the cells canbe very low, even <1%, because the synthesis occurs directly in thetargeted cells. Using different RNAs transcribed at different levels inthe same cells, it is possible change the amount of the proteinsynthesized by this method.

The variety of proteins which can be synthesized by the proposed methodis enormous. Limitations could occur if the proteins to be synthesisedare very large or have many hydrophobic amino acids.

The distance between the 5′ and 3′ ends of the oligomer-PAs afterhybridization to the template can be varied between 0 and 10 nucleotidesof the target RNA.

In the example described above, the oligomers are antiparallel to theplasminogen antigen activator mRNA. Using RNAs which expressedspecifically in different tumour cells, the synthesis of any protein inthese cells can be achieved. One example of such RNA is metastasin(mts-1) mRNA (Tulchinsky et al.1992, accession number g486654).

Using oligomers antiparallel to metastasin mRNA it is possible tosynthesise any toxin or protein specifically in human metastatic cells.

Using different RNAs expressed specifically in different tissues or incells infected by viruses, or in bacterial cells, it is possible tosynthesise any toxin or protein specifically in these cells.

THE EXAMPLE 10

Synthesis of the tumour suppresser p53 according to Formula 7. Afterhybridization of the oligomer-PAs to mRNA specific to ovarian tumourcells (NbHOT Homo sapiens mRNA accession number AA402345), the chemicalmoiety K̂1 of PA₂ (in this example K̂1 is NH₂ group) interacts with thelinking moiety L̂2 of the oligomer₁-PA₁. After the interaction hasoccurred, the peptide PA₁ is bound through the peptidyl bond to thepeptide PA₂ and is released from the 5′ end of the oligomer₁. Thelinking moiety L̂2 of the oligomer₁ is activated so that it interactswith the linking moiety L̂1 of oligomer₂, and the peptide PA₁-C(O)NH-PA₂is released from the 3′ end of oligomer₂. The chemical moiety K̂1 ofoligomer₃-PA₃ interacts with the linking moiety L̂2 ofoligomer₂-{PA₁-C(O)NH-PA₂} to bind peptide PA₃ with PA₁-C(O)NH-PA₂,releasing peptide PA₁-C(O)NH-PA₂-C(O)NH-PA₃ from oligomer₂. Theactivated linking moiety L̂2 of oligomer₂ interacts with the linkingmoiety L̂1 and releases the peptide PA₁-C(O)NH-PA₂-C(O)NH-PA₃ from the 3′ends of oligomer₃. The processes described above are repeated in thecells 13 times. In such as manner, the protein:{PA₁-C(O)NH-PA₂-C(O)NH-PA₃-C(O)NH-PA₄-C(O)NH-PA₅-C(O)NH-PA₆-C(O)NH-PA₇-C(O)NH-PA₈-C(C)NH-PA₉-C(O)NH-PA₁₀-C(O)NH-PA₁₁-C(O)NH-PA₁₂-C(O)NH-PA₁₃-C(O)NH-PA₁₄}can be synthesized. Neither the degradation of the oligomers nor thedegradation of the linking moieties is necessary to release the proteinfrom the oligomers. Peptidyl bond formation between Pa_(n-1) and PA_(n)and degradation of the linking moieties L̂2 proceed simultaneously withthe release of PAs from the 5′ ends of the oligomers. The activatedlinking moieties L̂2 interact with the linking moieties L̂1 to release thebound peptides from the 3′ ends of the oligomers.

PA₁—HEEPQSDPSVEPPLSOETFSDLWKLLPENNVL (SEQ ID No. 3)

PA₂—SPLPSQAMDDLMLSPDDIEQWF (SEQ ID No. 4)

PA₃—TEDPGPDEAPRMPEAAPRVAPAPMP (SEQ ID No. 5)

PA₄—TPMPAPAPSWPLSSSVPSQKJYQG (SEQ ID No. 6)

PA₅—SYGFRLGFLHSGTAKSVTCTY (SEQ ID No. 7)

PA₆—SPALNKMFCQLAKTCPVQLWVDSTPPPG (SEQ ID No. 8)

PA₇—TRVRAMAIYKQSQHMTEVVRRCPHHE (SEQ ID No. 9)

PA₈—TCSDSOGLAPPQHLIRVEGNLRVEYLDDRN (SEQ ID No. 10)

PA₉—TFRHSVVVPYEPPEVGSDCTTIHYNYMCNS (SEQ ID No. 11)

PA₁₀—SCMGGMNRRPIL TIITLEDSSGNLLGRN (SEQ ID No. 12)

PA₁₁—SFEVRVCACPGRORRTEEENLRKKGEPHHELPPG (SEQ ID No. 13)

PA₁₂—TKRALPNNTSSSPQPKKKPLDGEYF (SEQ ID No. 14)

PA₁₃—TLQIRGRERFEMFRELNEALELKDAQAGKEPGG (SEQ ID No. 15)

PA₁₄—SRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID No. 16)

where PA₁ to PA₁₄ are peptides bound to oligomers,

Aminoacids are designated in bold/italicised one letter code.

A—alanine, R—arginine, N—asparagine, D—aspartic acid, C—cysteine,Q—glutamine, E—glutamic acids, G—glycine, H—histidine, I—isoleucine,L—leucine, K—lysine, M—methionine, F—phenylalanine, P—proline, S—serine,T—threonine, W—tryptophan, Y—tyrosine, V—valine.

Oligomer₁ 3′ ATGGGCGGTAGGTAC 5′ (SEQ ID No. 31) Oligomer₂3′ TAGCGGTGCCCTCGA 5′ (SEQ ID No. 32) Oligomer₃ 3′ AACCCCGACGTCACG 5′(SEQ ID No. 33) Oligomer₄ 3′ TTCCGGACCCACGGA 5′ (SEQ ID No. 34)Oligomer₅ 3′ CGAGGTACAGGCCCC 5′ (SEQ ID No. 35) Oligomer₆3′ TACTCGAGTGTCTCG 5′ (SEQ ID No. 36) Oligomer₇ 3′ ACGACCGTCCCTAGT 5′(SEQ ID No. 37) Oligomer₈ 3′ GACCGTGACTTCACC 5′ (SEQ ID No. 38)Oligomer₉ 3′ TGACGGACGCCCGGA 5′ (SEQ ID No. 39) Oligomer₁₀3′ CAGTCCTCGTCTAGC 5′ (SEQ ID No. 40) Oligomer₁₁ 3′ TTCGACGTGAGTCCC 5′(SEQ ID No. 41) Oligomer₁₂ 3′ TCTCGGAGTCCCTTC 5′ (SEQ ID No. 42)Oligomer₁₃ 3′ GGAGAGTCTGGTCGA 5′ (SEQ ID No. 43) Oligomer₁₄3′ GGTCGGGTCGCGGGT 5′ (SEQ ID No. 44)

Oligomers are complementary (antiparallel) to NbHOT Homo sapiens mRNA(clone 741045 accession number AA402345) which is specific to ovariantumour cells. The distance of the oligomers each from other is nuNnucleotides of the NbHOT Homo sapiens mRNA.

Oligomer₁-PA₁ is MEEPQSDPSVEPPLSQETFSDLWKLLPENNVL (SEQ ID No. 3)                               L{circumflex over ( )}23′ ATGGGCGGTAGGTAC 5′ (SEQ ID No. 31) Oligomer₂-PA₂ is (K{circumflexover ( )}1)SPLPSQAMDDLMLSPDDIEQWF (SEQ ID No. 4)      L{circumflex over( )}1                  L{circumflex over ( )}2 3′ TAGCGGTGCCCTCGA 5′(SEQ ID No. 32) Oligomer₃-PA₃ is (K{circumflex over( )}1)TEDPGPDEAPRMPEAAPRVAPAPAAP (SEQ ID No. 5)       L{circumflex over( )}1                     L{circumflex over ( )}2 3′ AACCCCGACGTCACG 5′(SEQ ID No. 33) Oligomer₄-PA₄ is (K{circumflex over( )}1)TPAAPAPAPSWPLSSSVPSQKTYQG (SEQ ID No. 6)       L{circumflex over( )}1                    L{circumflex over ( )}2 3′ TTCCGGACCCACGGA 5′(SEQ ID No. 34) Oligomer₅-PA₅ is (K{circumflex over( )}1)SYGFRLGFLHSGTAKSVTCTY (SEQ ID No. 7)       L{circumflex over( )}1                L{circumflex over ( )}2 3′ CGAGGTACAGGCCCC 5′ (SEQID No. 35) Oligomer₆-PA₆ is (K{circumflex over( )}1)SPALNKMFCQLAKTCPVQLWVDSTPPPG (SEQ ID No. 8)       L{circumflexover ( )}1                       L{circumflex over ( )}2 3TACTCGAGTGTCTCG 5′ (SEQ ID No. 36) Oligomer₇-PA₇ is (K{circumflex over( )}1)TRVRAMAIYKOSQHMTEVVRRCPHHE (SEQ ID No. 9)       L{circumflex over( )}1                     L{circumflex over ( )}2 3′ ACGACCGTCCCTAGT 5′(SEQ ID No. 37) Oligomer₈-PA₈ is (K{circumflex over( )}1)TCSDSDGLAPPQHLIRVEGNLRVEYLDDRRN (SEQ ID No. 10)       L{circumflexover ( )}1                         L{circumflex over ( )}23′ GACCGTGACTTCACC 5′ (SEQ ID No. 38) Oligomer₉-PA₉ is (K{circumflexover ( )}1)TFRHSVVVPYEPPEVGSDCTTIHYNYMCNS (SEQ ID No. 11)      L{circumflex over ( )}1                        L{circumflex over( )}2 3′ TGACGGACGCCCGGA 5′ (SEQ ID No. 39) Oligomer₁₀-PA₁₀ is(K{circumflex over ( )}1)SCMGGMNRRPILTIITLEDSSGNLLGRNS (SEQ ID No. 12)     L{circumflex over ( )}1                         L{circumflex over( )}2 3′ CAGTCCTCGTCTAGC 5′ (SEQ ID No. 40) Oligomer₁₁-PA₁₁ is(K{circumflex over ( )}1)FEVRVCACPGRDRRTEEENLRKKGEPHHELPPGS (SEQ ID No.13)       L{circumflex over ( )}1                           L{circumflexover ( )}2 3′ TTCGACGTGAGTCCC 5′ (SEQ ID No. 41) Oligomer₁₂-PA₁₂ is(K{circumflex over ( )}1)TKRALPNNTSSSPQPKKKPLDGEYF (SEQ ID No. 14)      L{circumflex over ( )}1                   L{circumflex over ( )}23′ TCTCGGAGTCCCTTC5′ (SEQ ID No. 42) Oligomer₁₃-PA₁₃ is (K{circumflexover ( )}1)TLQIRGRERFEMFRELNEALELKDAQAGKEPGG (SEQ ID No. 15)      L{circumflex over ( )}1                           L{circumflexover ( )}2 3′ GGAGAGTCTGGTCGA 5′ (SEQ ID No. 43) Oligomer₁₄-PA₁₄ is(K{circumflex over ( )}1)SRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSD (SEQ ID No.16)      L{circumflex over ( )}1 3′ GGTCGGGTCGCGGGT 5′ (SEQ ID No. 44)

This method of protein synthesis also allows modification of thesynthesized protein. Certain aminoacids of the peptides used in thesynthesis can be glycosylated or phosphorylated.

Glycosylation of a protein is a complex process, and difficulties mayoccur in the penetrance of some tissues with the glycosylated form ofthe peptide due to the size of the molecule.

However the use of phosphorylated peptides opens up the possibility tosynthesize already active proteins in the cells of living organisms.

The Synthesis of RNA.

Using the method described above, it is possible to synthesise intotargeted cells not only proteins but also RNAs. An example of suchsynthesis is represented in FIG. 10

To synthesize whole RNA in cells from n oligomers bound tooligoribonucleotides (oligomer-PAs) the concentration of sucholigomer-PAs must be high. After the simultaneous hybridization ofoligomer-PAs to the same molecule of the cellular RNA, the chemicallyactive 3′ hydroxyl group of the oligoribonucleotid PA₁ interacts withthe linking moiety -L̂2-S— which bound oligoribonucleotide PA₂ witholigomer 2. In this case the linking group is represented with a n—S-L̂2-moiety which is coupled to phosphate group of theoligoribonucleotide PA₂. The 3′ hydroxyl group of theoligoribonucleotide PA₁ interacts with the linking group of PA₂ forminga chemical bond with the phosphate group, releasing theoligoribonucleotide PA₂ at it's 5′ end from oligomer 2, and activatingthe linking moiety with the formation of the —SH group. This chemicallyactive group —SH interacts with linking moiety -L̂1-S which couples theoligomers. This process is repeated n-1 times to bind all PAs in onemolecule. PA₁ is bound through chemical moiety —O— to PA₂, then in turnPA₁-m-PA₂ is bound through chemical moiety —O— to PA₃, thenPA₁-m-PA₂-m-PA₃ is bound through chemical moiety —O— to PA₄ and so onuntil the last oligoribonucleotide is bound, forming whole biologicallyactive RNA.

In this figure “PA_(n)” are oligoribonucleotides comprising from 3 to300 nucleotides.

n in “PA_(n)” means the ordinal number in a series ofoligoribonucleotides used in the synthesis of a whole RNA, where n isselected from 2 to 1000.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 44 <210> SEQ ID NO 1 <211>LENGTH: 16 <212> TYPE: DNA <213> ORGANISM: Human immunodeficiency virustype 1 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSION NUMBER:X01762 <309> DATABASE ENTRY DATE: 1985-01-01 <310> PATENT DOCUMENTNUMBER: US 5,571,937 <311> PATENT FILING DATE: 1994-05-13 <312>PUBLICATION DATE: 1996-01-11 <313> RELEVANT RESIDUES: (1)..(16) <400>SEQUENCE: 1 gaaggaatag aagaag 16 <210> SEQ ID NO 2 <211> LENGTH: 16<212> TYPE: DNA <213> ORGANISM: Human immunodeficiency virus type 1<300> PUBLICATION INFORMATION: <302> TITLE: Complementary DNA and Toxins(seq ID 43) <308> DATABASE ACCESSION NUMBER: X01762 <309> DATABASE ENTRYDATE: 1985-01-01 <310> PATENT DOCUMENT NUMBER: US 5,571,937 <311> PATENTFILING DATE: 1994-05-13 <312> PUBLICATION DATE: 1996-05-11 <313>RELEVANT RESIDUES: (17)..(32) <400> SEQUENCE: 2 aaggtggaga gagaga 16<210> SEQ ID NO 3 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: part of the amino acidsequence of the Human tumour supressor p53 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Harlow,E., Williamson,N.M., Ralston,R.,Helfman,D.M. and Adams,T.E. <302> TITLE: Molecular cloning and in vitroexpression of a cDNA clone for human cellular tumor antigen p53 <303>JOURNAL: Molecular and cellular biology <304> VOLUME: 5 <305> ISSUE: 7<306> PAGES: 1601-1610 <307> DATE: 1985-07-01 <308> DATABASE ACCESSIONNUMBER: K03199 <309> DATABASE ENTRY DATE: 1995-01-07 <400> SEQUENCE: 3Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln 1 5 1015 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu 20 2530 <210> SEQ ID NO 4 <211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: part of the aminoacid sequence of the Human tumour supressor p53 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Harlow,E., Williamson,N.M., Ralston,R.,Helfman,D.M. and Adams,T.E. <302> TITLE: Molecular cloning and in vitroexpression of a cDNA clone for human cellular tumor antigen p53 <303>JOURNAL: Molecular and cellular biology <304> VOLUME: 5 <305> ISSUE: 7<306> PAGES: 1601-10 <307> DATE: 1985-06-01 <308> DATABASE ACCESSIONNUMBER: K03199 <309> DATABASE ENTRY DATE: 1995-01-07 <400> SEQUENCE: 4Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp 1 5 1015 Asp Ile Glu Gln Trp Phe 20 <210> SEQ ID NO 5 <211> LENGTH: 26 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHERINFORMATION: part of the amino acid sequence of the Human tumoursupressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS: Harlow,E.,Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E. <302> TITLE:Molecular cloning and in vitro expression of a cDNA clone for humancellular tumor antigen p53 <303> JOURNAL: Molecular and cellular biology<304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10 <307> DATE:1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309> DATABASE ENTRYDATE: 1995-07-01 <400> SEQUENCE: 5 Thr Glu Asp Pro Gly Pro Asp Glu AlaPro Arg Met Pro Glu Ala Ala 1 5 10 15 Pro Arg Val Ala Pro Ala Pro AlaAla Pro 20 25 <210> SEQ ID NO 6 <211> LENGTH: 25 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: part ofthe amino acid sequence of the Human tumour supressor p53 <300>PUBLICATION INFORMATION: <301> AUTHORS: Harlow,E., Williamson,N.M.,Ralston,R., Helfman,D.M. and Adams,T.E. <302> TITLE: Molecular cloningand in vitro expression of a cDNA clone for human cellular tumor antigenp53 <303> JOURNAL: Molecular and cellular biology <304> VOLUME: 5 <305>ISSUE: 7 <306> PAGES: 1601-10 <307> DATE: 1985-01-07 <308> DATABASEACCESSION NUMBER: K03199 <309> DATABASE ENTRY DATE: 1995-07-01 <400>SEQUENCE: 6 Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser SerSer 1 5 10 15 Val Pro Ser Gln Lys Thr Tyr Gln Gly 20 25 <210> SEQ ID NO7 <211> LENGTH: 21 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: part of the amino acid sequence of theHuman tumour supressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS:Harlow,E., Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E.<302> TITLE: Molecular cloning and in vitro expression of a cDNA clonefor human cellular tumor antigen p53 <303> JOURNAL: Molecular andcellular biology <304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10<307> DATE: 1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309>DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 7 Ser Tyr Gly Phe ArgLeu Gly Phe Leu His Ser Gly Thr Ala Lys Ser 1 5 10 15 Val Thr Cys ThrTyr 20 <210> SEQ ID NO 8 <211> LENGTH: 28 <212> TYPE: PRT <213>ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: part ofthe amino acid sequence of the Human tumour supressor p53 <300>PUBLICATION INFORMATION: <301> AUTHORS: Harlow,E., Williamson,N.M.,Ralston,R., Helfman,D.M. and Adams,T.E. <302> TITLE: Molecular cloningand in vitro expression of a cDNA clone for human cellular tumor antigenp53 <303> JOURNAL: Molecular and cellular biology <304> VOLUME: 5 <305>ISSUE: 7 <306> PAGES: 1601-10 <307> DATE: 1985-01-07 <308> DATABASEACCESSION NUMBER: K03199 <309> DATABASE ENTRY DATE: 1995-07-01 <400>SEQUENCE: 8 Ser Pro Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr CysPro 1 5 10 15 Val Gln Leu Trp Val Asp Ser Thr Pro Pro Pro Gly 20 25<210> SEQ ID NO 9 <211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: part of the amino acidsequence of the Human tumour supressor p53 <300> PUBLICATIONINFORMATION: <301> AUTHORS: Harlow,E., Williamson,N.M., Ralston,R.,Helfman,D.M. and Adams,T.E. <302> TITLE: Molecular cloning and in vitroexpression of a cDNA clone for human cellular tumor antigen p53 <303>JOURNAL: Molecular and cellular biology <304> VOLUME: 5 <305> ISSUE: 7<306> PAGES: 1601-10 <307> DATE: 1985-01-07 <308> DATABASE ACCESSIONNUMBER: K03199 <309> DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 9Thr Arg Val Arg Ala Met Ala Ile Tyr Lys Gln Ser Gln His Met Thr 1 5 1015 Glu Val Val Arg Arg Cys Pro His His Glu 20 25 <210> SEQ ID NO 10<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: part of the amino acid sequence of theHuman tumour supressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS:Harlow,E., Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E.<302> TITLE: Molecular cloning and in vitro expression of a cDNA clonefor human cellular tumor antigen p53 <303> JOURNAL: Molecular andcellular biology <304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10<307> DATE: 1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309>DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 10 Thr Cys Ser Asp SerAsp Gly Leu Ala Pro Pro Gln His Leu Ile Arg 1 5 10 15 Val Glu Gly AsnLeu Arg Val Glu Tyr Leu Asp Asp Arg Asn 20 25 30 <210> SEQ ID NO 11<211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: part of the amino acid sequence of theHuman tumour supressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS:Harlow,E., Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E.<302> TITLE: Molecular cloning and in vitro expression of a cDNA clonefor human cellular tumor antigen p53 <303> JOURNAL: Molecular andcellular biology <304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10<307> DATE: 1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309>DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 11 Thr Phe Arg His SerVal Val Val Pro Tyr Glu Pro Pro Glu Val Gly 1 5 10 15 Ser Asp Cys ThrThr Ile His Tyr Asn Tyr Met Cys Asn Ser 20 25 30 <210> SEQ ID NO 12<211> LENGTH: 28 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: part of the amino acid sequence of theHuman tumour supressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS:Harlow,E., Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E.<302> TITLE: Molecular cloning and in vitro expression of a cDNA clonefor human cellular tumor antigen p53 <303> JOURNAL: Molecular andcellular biology <304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10<307> DATE: 1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309>DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 12 Ser Cys Met Gly GlyMet Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr 1 5 10 15 Leu Glu Asp SerSer Gly Asn Leu Leu Gly Arg Asn 20 25 <210> SEQ ID NO 13 <211> LENGTH:34 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <223>OTHER INFORMATION: part of the amino acid sequence of the Human tumoursupressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS: Harlow,E.,Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E. <302> TITLE:Molecular cloning and in vitro expression of a cDNA clone for humancellular tumor antigen p53 <303> JOURNAL: Molecular and cellular biology<304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10 <307> DATE:1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309> DATABASE ENTRYDATE: 1995-07-01 <400> SEQUENCE: 13 Ser Phe Glu Val Arg Val Cys Ala CysPro Gly Arg Asp Arg Arg Thr 1 5 10 15 Glu Glu Glu Asn Leu Arg Lys LysGly Glu Pro His His Glu Leu Pro 20 25 30 Pro Gly <210> SEQ ID NO 14<211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: part of the amino acid sequence of theHuman tumour supressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS:Harlow,E., Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E.<302> TITLE: Molecular cloning and in vitro expression of a cDNA clonefor human cellular tumor antigen p53 <303> JOURNAL: Molecular andcellular biology <304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10<307> DATE: 1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309>DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 14 Ser Thr Lys Arg AlaLeu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro 1 5 10 15 Lys Lys Lys ProLeu Asp Gly Glu Tyr Phe 20 25 <210> SEQ ID NO 15 <211> LENGTH: 33 <212>TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHERINFORMATION: part of the amino acid sequence of the Human tumoursupressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS: Harlow,E.,Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E. <302> TITLE:Molecular cloning and in vitro expression of a cDNA clone for humancellular tumor antigen p53 <303> JOURNAL: Molecular and cellular biology<304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10 <307> DATE:1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309> DATABASE ENTRYDATE: 1995-07-01 <400> SEQUENCE: 15 Thr Leu Gln Ile Arg Gly Arg Glu ArgPhe Glu Met Phe Arg Glu Leu 1 5 10 15 Asn Glu Ala Leu Glu Leu Lys AspAla Gln Ala Gly Lys Glu Pro Gly 20 25 30 Gly <210> SEQ ID NO 16 <211>LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:<223> OTHER INFORMATION: part of the amino acid sequence of the Humantumour supressor p53 <300> PUBLICATION INFORMATION: <301> AUTHORS:Harlow,E., Williamson,N.M., Ralston,R., Helfman,D.M. and Adams,T.E.<302> TITLE: Molecular cloning and in vitro expression of a cDNA clonefor human cellular tumor antigen p53 <303> JOURNAL: Molecular andcellular biology <304> VOLUME: 5 <305> ISSUE: 7 <306> PAGES: 1601-10<307> DATE: 1985-01-07 <308> DATABASE ACCESSION NUMBER: K03199 <309>DATABASE ENTRY DATE: 1995-07-01 <400> SEQUENCE: 16 Ser Arg Ala His SerSer His Leu Lys Ser Lys Lys Gly Gln Ser Thr 1 5 10 15 Ser Arg His LysLys Leu Met Phe Lys Thr Glu Gly Pro Asp Ser Asp 20 25 30 <210> SEQ ID NO17 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: antisense to the human tissueplasminogen activator mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS:Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The human tissueplasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 17 cccaatccct cttgcaactg a 21<210> SEQ ID NO 18 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 18 attctactac aagtctgccc tt 22<210> SEQ ID NO 19 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 19 ttgtgaccgg ctccactg 18 <210>SEQ ID NO 20 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 20 taccttggta cttctctaa 19 <210>SEQ ID NO 21 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 21 atgccatatt agcccatcag a 21<210> SEQ ID NO 22 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 22 ccaagcattc tgtccctcct tt 22<210> SEQ ID NO 23 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 23 tccggtccgg agcacca 17 <210>SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 24 gccatgacct gtatgttaca 20 <210>SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 25 ggtgtgggaa agttagcggg 20 <210>SEQ ID NO 26 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 26 gcgaattcca aatgatttta a 21<210> SEQ ID NO 27 <211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 27 aatgtgaaca tgaataa 17 <210>SEQ ID NO 28 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 28 agagtgggat acagcatcta ta 22<210> SEQ ID NO 29 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 29 acaaaaccat tccactctga tt 22<210> SEQ ID NO 30 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: antisense to the humantissue plasminogen activator mRNA <300> PUBLICATION INFORMATION: <301>AUTHORS: Degen,S.J., Rajput,B. and Reich,E. <302> TITLE: The humantissue plasminogen activator gene <303> JOURNAL: Journal of BiologicalChemistry <304> VOLUME: 261 <305> ISSUE: 15 <306> PAGES: 6972-85 <307>DATE: 1986-05-25 <308> DATABASE ACCESSION NUMBER: K03021 <309> DATABASEENTRY DATE: 1986-04-08 <400> SEQUENCE: 30 ttggaaaaac tgtgaaaaa 19 <210>SEQ ID NO 31 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homosapiens <220> FEATURE: <223> OTHER INFORMATION: Antisense to the humanNbHOT (cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS:Inaba,T., Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. andLook,A.T <302> TITLE: Genomic organization, chromosomal localization,and independent expression of human cyclin D genes <303> JOURNAL:Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306> PAGES: 565-574 <307>DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER: NM_001760 <309>DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATION INFORMATION: <308>DATABASE ACCESSION NUMBER: AA402345 <309> DATABASE ENTRY DATE:1996-12-20 <400> SEQUENCE: 31 catggatggc gggta 15 <210> SEQ ID NO 32<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220>FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT (cyclinD) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:32 agctcccgtg gcgat 15 <210> SEQ ID NO 33 <211> LENGTH: 15 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:Antisense to the human NbHOT (cyclin D) mRNA <300> PUBLICATIONINFORMATION: <301> AUTHORS: Inaba,T., Matsushime,H., Valentine,M.,Roussel,M.F., Sherr,C.J. and Look,A.T <302> TITLE: Genomic organization,chromosomal localization, and independent expression of human cyclin Dgenes <303> JOURNAL: Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306>PAGES: 565-574 <307> DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER:NM_001760 <309> DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: AA402345 <309> DATABASEENTRY DATE: 1996-12-20 <400> SEQUENCE: 33 gcactgcagc cccaa 15 <210> SEQID NO 34 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT(cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:34 aggcacccag gcctt 15 <210> SEQ ID NO 35 <211> LENGTH: 15 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:Antisense to the human NbHOT (cyclin D) mRNA <300> PUBLICATIONINFORMATION: <301> AUTHORS: Inaba,T., Matsushime,H., Valentine,M.,Roussel,M.F., Sherr,C.J. and Look,A.T <302> TITLE: Genomic organization,chromosomal localization, and independent expression of human cyclin Dgenes <303> JOURNAL: Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306>PAGES: 565-574 <307> DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER:NM_001760 <309> DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: AA402345 <309> DATABASEENTRY DATE: 1996-12-20 <400> SEQUENCE: 35 ccccggacat ggagc 15 <210> SEQID NO 36 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT(cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:36 gctctgtgag ctcat 15 <210> SEQ ID NO 37 <211> LENGTH: 15 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:Antisense to the human NbHOT (cyclin D) mRNA <300> PUBLICATIONINFORMATION: <301> AUTHORS: Inaba,T., Matsushime,H., Valentine,M.,Roussel,M.F., Sherr,C.J. and Look,A.T <302> TITLE: Genomic organization,chromosomal localization, and independent expression of human cyclin Dgenes <303> JOURNAL: Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306>PAGES: 565-574 <307> DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER:NM_001760 <309> DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: AA402345 <309> DATABASEENTRY DATE: 1996-12-20 <400> SEQUENCE: 37 tgatccctgc cagca 15 <210> SEQID NO 38 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT(cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:38 ccacttcagt gccag 15 <210> SEQ ID NO 39 <211> LENGTH: 15 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:Antisense to the human NbHOT (cyclin D) mRNA <300> PUBLICATIONINFORMATION: <301> AUTHORS: Inaba,T., Matsushime,H., Valentine,M.,Roussel,M.F., Sherr,C.J. and Look,A.T <302> TITLE: Genomic organization,chromosomal localization, and independent expression of human cyclin Dgenes <303> JOURNAL: Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306>PAGES: 565-574 <307> DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER:NM_001760 <309> DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: AA402345 <309> DATABASEENTRY DATE: 1996-12-20 <400> SEQUENCE: 39 aggcccgcag gcagt 15 <210> SEQID NO 40 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT(cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:40 cgatctgctc ctgac 15 <210> SEQ ID NO 41 <211> LENGTH: 15 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:Antisense to the human NbHOT (cyclin D) mRNA <300> PUBLICATIONINFORMATION: <301> AUTHORS: Inaba,T., Matsushime,H., Valentine,M.,Roussel,M.F., Sherr,C.J. and Look,A.T <302> TITLE: Genomic organization,chromosomal localization, and independent expression of human cyclin Dgenes <303> JOURNAL: Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306>PAGES: 565-574 <307> DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER:NM_001760 <309> DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: AA402345 <309> DATABASEENTRY DATE: 1996-12-20 <400> SEQUENCE: 41 ccctgagtgc agctt 15 <210> SEQID NO 42 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT(cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:42 cttccctgag gctct 15 <210> SEQ ID NO 43 <211> LENGTH: 15 <212> TYPE:DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION:Antisense to the human NbHOT (cyclin D) mRNA <300> PUBLICATIONINFORMATION: <301> AUTHORS: Inaba,T., Matsushime,H., Valentine,M.,Roussel,M.F., Sherr,C.J. and Look,A.T <302> TITLE: Genomic organization,chromosomal localization, and independent expression of human cyclin Dgenes <303> JOURNAL: Genomics <304> VOLUME: 13 <305> ISSUE: 3 <306>PAGES: 565-574 <307> DATE: 1992-07-01 <308> DATABASE ACCESSION NUMBER:NM_001760 <309> DATABASE ENTRY DATE: 2001-11-16 <300> PUBLICATIONINFORMATION: <308> DATABASE ACCESSION NUMBER: AA402345 <309> DATABASEENTRY DATE: 1996-12-20 <400> SEQUENCE: 43 agctggtctg agagg 15 <210> SEQID NO 44 <211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Homo sapiens<220> FEATURE: <223> OTHER INFORMATION: Antisense to the human NbHOT(cyclin D) mRNA <300> PUBLICATION INFORMATION: <301> AUTHORS: Inaba,T.,Matsushime,H., Valentine,M., Roussel,M.F., Sherr,C.J. and Look,A.T <302>TITLE: Genomic organization, chromosomal localization, and independentexpression of human cyclin D genes <303> JOURNAL: Genomics <304> VOLUME:13 <305> ISSUE: 3 <306> PAGES: 565-574 <307> DATE: 1992-07-01 <308>DATABASE ACCESSION NUMBER: NM_001760 <309> DATABASE ENTRY DATE:2001-11-16 <300> PUBLICATION INFORMATION: <308> DATABASE ACCESSIONNUMBER: AA402345 <309> DATABASE ENTRY DATE: 1996-12-20 <400> SEQUENCE:44 tgggcgctgg gctgg 15

What is claimed is:
 1. A process for synthesis of biologically activecompounds from biologically inactive precursors with the aim to deliversaid biologically active compounds to the target cells having specificRNA or DNA molecules, the process comprising: using a compositioncomprising at least two different oligomers chemically bound at their 5′and/or 3′ end via a linking moiety to said biologically inactiveprecursors of biologically active compound, and said biologicallyinactive precursors comprising chemically active groups wherein saidoligomers allow the hybridisation to a cellular RNA, DNA or dsDNA suchthat after hybridisation the distance between the 3′ or 5′ ends of saidtwo oligomers coupled to the biologically inactive precursors is in therange of 0 to 8 ribo(deoxy)nucleotides and said hybridisation leads to achemical coupling of said two precursors via said chemically activegroups and/or said linking moieties and the intracellular synthesis ofsaid biologically active compound in the target cells.
 2. The process ofclaim 1, wherein said chemically active group of said biologicallyinactive precursor is selected from the group consisting of —NH(2)-,—NH—, —OH, —SH, —F, —CL, —Br, —I, and —R̂1-C(X)—X̂1-R̂2, wherein R̂1 and R̂2are independently chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X̂1-P(X)(X)—X̂1, —S(O)—, —S(O)(O)—, —X̂1,—C(O)—, —N(H)—, —N═N—, —X̂1-P(X)(X)—X̂1-, —X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1,—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1, —C(s)—, any suitable linkinggroup: wherein X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl:X̂1 is independently S, O, NH, Se, alkyl, alkenyl, alkynyl.
 3. Theprocess of claim 1, wherein said at least two biologically inactiveprecursors are coupled via a chemical bond selected from the groupconsisting of: —S—S—, —O—, —NH—C(O)—, —C(O)—NH—, —C(O)O—, —C(O)—S—, —S—,—C(S)S—, —C(S)O—.
 4. The process of claim 1, wherein said oligomers areselected from the group consisting of oligomers 2 to
 14. 5. The processof claim 1, wherein said linking moiety connecting said oligomer to saidbiologically inactive precursor is selected from the group consistingof: L̂2 is independently: chemical bond, —R̂1-, —R̂1-O—S—R̂2-, —R̂1-S—O—R̂2-,R̂1-S—S—R̂2-, R̂1-S—N(H)—R̂2-, —R̂1-N(H)—S—R̂2-, —R̂1-O—N(H)—R̂2-,R̂1-N(H)—O—R̂2-, —R̂1-C(X)—X̂1-R̂2-, —R̂1-X—C(X)—X—C(X)—X—R̂2-; wherein R̂1 isindependently: chemical bond, alkyl, alkenyl, alkynyl, aryl,heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, cycloheteroaryl,carbocyclic, heterocyclic ring, X̂1-P(X)(X)—X̂1, —S(O)—, —S(O)(O)—, −111X̂1-S(X)(X)—X̂1-, —C(O)—, —N(H)—, —N═N—, —X̂1-P(X)(X)—X̂1-,—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1-, —X̂1-P(X)(X)—X̂1, -P(X)(X)—X̂1-P(X)(X)—X̂1,—C(S)—, any suitable linking group: wherein R̂2 is independently chemicalbond, alkyl, alkenyl, alkynyl, aryl, heteroalkyl, heteroalkenyl,heteroalkynyl, heteroaryl, cycloheteroaryl, carbocyclic, heterocyclicring, X̂1-P(X)(X)—X̂1, —S(O)—, S(O)(O)—, —X̂1-S(X)(X)—X̂1, —C(O)—, —N(H)—,—N═N—, —X̂1-P(X)(X)—X̂1-, —X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1,—X̂1-P(X)(X)—X̂1-P(X)(X)—X̂1 -P(X)(X)—X̂1 , —C(S)—, any suitable linkinggroup: wherein X is independently S, O, NH, Se, alkyl, alkenyl, alkynyl:X̂1 is independently S. 0, NH, Se. alkyl, alkenyl. alkynyl.
 6. Theprocess of claim 1, wherein the said biologically inactive precursorsare peptides comprising from 2 to 100 aminoacids.
 7. The process ofclaim 1, wherein said biologically active compound is selected from thegroup consisting of: proteins, enzymes, peptides, cyclic peptides, toxicpeptides, modified toxic peptides and toxic proteins.
 8. The process ofclaim 1, wherein said biologically active compound is daphnoretin,amanitin, D-actinomicin, ochratoxin A, or ergotamin.